The normal functioning of the eukaryotic cell requires that all newly synthesized proteins be correctly folded, modified, and delivered to specific inter and extracellular sites. Newly synthesized membrane and secretory proteins enter a cellular sorting and distribution network during or immediately after synthesis (cotranslationally or posttranslationally) and are routed to specific locations inside and outside of the cell. The initial compartment in this process is the endoplasmic reticulum (ER) where proteins undergo modifications such as glycosylation, disulfide bond formation, and assembly into oligomers. The proteins are then transported through an additional series of membrane-bound compartments which include the various cisternae of the Golgi complex, where further carbohydrate modifications occur. Transport between compartments occurs by means of vesicles that bud and fuse in a specific manner; once within the secretory pathway, proteins do not have to cross a membrane to reach the cell surface.
The complexity of this system has advantages for the cell because it allows proteins to fold and mature in closed compartments that contain the appropriate enzyme catalysts. It is, however, dependent on sorting mechanisms that position the enzymes correctly and maintain them in place.
The first organelle in this system, the ER, contains multiple enzymes involved in protein structure modifications. Among these are BiP (binding protein) which directs the correct folding of proteins and, PDI (protein disulfide isomerase) and a homologue of the 90 kDa heat-shock protein, both of which catalyze the formation and rearrangement of disulfide bonds (Gething, M. J. and Sambrook, J. (1992) Nature 355:33-45). These abundant soluble proteins must be retained in the ER and must be distinguished from the newly synthesized secretory proteins which are rapidly transported to the Golgi apparatus. The signal for retention in the ER in mammalian cells consists of the tetrapeptide sequence, KDEL, located at the carboxy terminus of proteins. This sequence was first identified when the sequences of rat BiP and PDI were compared and it was subsequently found at the carboxy terminus of other luminal ER proteins from a number of species (Munro, S. (1986) Cell 46:291-300; Pelham, H. R. (1989) Ann. Rev. Cell. Biol. 5:1-23). Proteins containing this sequence leave the ER but are quickly retrieved from the early Golgi compartment and returned to the ER, while proteins without this signal continue through the distribution pathway.
Two endoplasmic retrieval receptors were first identified in S. cerevesiae; two human endoplasmic retrieval receptors were subsequently isolated by the use of degenerate PCR primers based on the S. cerevesiae sequences (Hardwick, K. G. (1990) EMBO J. 9:623-630; Semenza, J. C. (1990) Cell 61:1349-1357; Lewis, M. J. and Pelham, H. R. (1990) Nature 348:162-163; Lewis, M. J. and Pelham, H. R. (1992) J. Mol. Biol. 226:913-916). Comparisons of these sequences shows that they consist of a conserved 7-transmembrane domain structure with only short loops in the cell cytoplasm and the ER lumen. Studies with these endoplasmic retrieval receptors show that ligand binding controls the movement of the receptor; when expressed in COS cells, the human receptor is normally concentrated in the Golgi, but moves to the ER when bound to a ligand such as KDEL-tagged hen lysozyme (Lewis, M. J. and Pelham, H. R. (1992) Cell 68:353-364).
The ER retrieval function of these molecules serves to maintain the pool of enzymes in the ER that are necessary to perform protein structure modifications, retains newly synthesized proteins in the ER until they have been correctly modified, and regulates the structure of the Golgi apparatus. Saccharomyces cerevisiae cells that lack an ER retrieval receptor (Erd2) have a defective Golgi apparatus and fail to grow. Analysis of yeast Erd2 mutants suggests that their growth requires both the retention of multiple proteins in the ER and the selective removal of specific proteins from the Golgi (Townsley, F. M. (1994) J. Cell Biol. 127:21-28). Overexpression of a human ER retrieval receptor in COS cells results in hyperactive retrograde traffic from the Golgi to the ER leading to a loss of the Golgi structure and the breakdown of the secretory pathway (Hsu V. W. (1992) Cell 69:625-635).
Disruptions in the cellular secretory pathway have been implicated in several human diseases. In familial hypercholesterolemia the low density lipoprotein receptors remain in the ER, rather than moving to the cell surface (Pathak, R. K. (1988) J. Cell Biol. 106:1831-1841). A form of congenital hypothyroidism is produced by a deficiency of thyroglobulin, the thyroid prohormone. In this disease the thyroglobulin is incorrectly folded and is therefore retained in the ER (Kim, P. S. (1996) J.Cell Biol. 133:517-527). Mutant forms of proteolipid protein (PLP) have been examined as they play a role in generating dysmyelinating or hypomyelinating diseases. In this case, the mutations that result in disease are mutations that arrest transport of PLP in the ER and the early Golgi; the subsequent accumulation of PLP in the ER results in rapid oligodendrocyte death (Gow, A. (1994) J. Neurosci. Res. 37:574-583).
The human ER retrieval receptor function is necessary for processing and presentation of specific antigens to T cells. Many antigens must be processed intracellularly before they can be presented, in association with major histocompatability complex (MHC) molecules at the cell surface, for recognition by the antigen-specific receptor of T cells. Disruption of the ER retrieval receptor function with an antibiotic, Brefeldin A, abolishes the ability of a cell to present these specific antigen complexes to T cells. These antigenic proteins must be retained in the ER for cleavage to smaller peptides which can then bind to MHC molecules and be released for presentation at the cell surface. (Kakiuchi, T. (1991) J. Immunol. 147:3289-3295).
The discovery of polynucleotides encoding a novel human KDEL receptor, and the molecules themselves, provides the means to further investigate the regulation of the cellular protein secretory pathway. Discovery of molecules related to a novel human KDEL receptor satisfies a need in the art by providing a means or a tool for the study of this pathway and the diseases that involve the dysfunction of this pathway.